The invention relates to a novel compound; to methods of diagnosis in medical and veterinary contexts using the compound; screening methods for therapeutic substances; and methods of treatment.
Biochemical markers in body fluids are valuable tools in clinical medicine. Biochemical tests are relevant in such diseases as diabetes, myocardial infarction, and osteoporosis. The objective of a biochemical marker is to detect latent disease, or to monitor pre-existing disease and its treatment. To date, there has been no useful marker found that is relevant to the assessment of cartilage in arthritic conditions.
Cartilage matrix is made up of a complex mixture of components including collagen type II, collagen IX and collagen XI, procollagen molecules, proteoglycans, aggrecan, hyaluronic acid, link proteins, etc. Several cartilage components have been studied for their clinical utility. The C-propeptide of type II collagen, a marker of type II collagen synthesis, may reflect cartilage repair (Poole AR, Cartilage in Health and Disease, In: Arthritis and Alllied Conditions. A Textbook of Rheumatology. 12th ed. McCarty D. Koopman W, editors. Malvern: Lea and Febiger, 1992:279-333, 1986). Another component, aggrecan, is usually elevated in osteoarthritic joint fluid (Ratcliffe, J. Oral Maxillofac Surg 49(7):708, 1991; Witter J, Arthritis Rheum 30(5):519, 1987). Some catabolic enzymes, especially metalloproteases, are found in osteoarthritic joint fluid (Walakovits, Arthritis Rheum. 35(1):35-42, 1992).
Fluorescent markers associated with bone disease are released from collagen and its peptide fragments through acid hydrolysis, and are relatively stable in biological fluids. The two known types of fluorescent compounds within cartilage, pyridinium compounds and pentosidine have been studied in synovial fluid, urine, blood, and other biological fluids. Pyridinium compound levels were found to be essentially unchanged with age, or decreased slightly, and pentosidine increased with age (Takahashi M et al. Arthritis and Rheumatism 37(5):724-728, 1994). Pyridinium compounds can be measured in the urine, serum, or plasma as a ratio with creatinine. In bone disease, pyridinium compounds are increased with bone resorption. In osteoarthritis and rheumatoid arthritis patients, urinary pyridinium compounds are increased. However, Robins (Biochem J. 207:617-620, 1992) concluded that the cartilage itself contributed very little to the urinary rise in pyridinium compounds in these patients and the rise was attributed to bone resorption rather than cartilage degradation.
A molecule that is chemically stable, can be measured in biological fluids, and which is specific to cartilage is desirable as a biochemical marker to aid in the assessment of, and monitoring the treatment of osteoarthritis and other arthritic conditions.
The present inventors have identified a novel compound derived from, and found in cartilage, articular cartilage containing tissues such as intervertebral disc, trachea, and sternum, and cartilagenous metaplasia and neoplasia of tissues. The novel compound was also detected in the synovial fluids of human and calf. The compound is also found in cartilage cultured in vitro. Anatomically, the level of the compound increases with the cartilage depth, and in this respect it parallels the proteoglycan content.
The present inventors observed that levels of the compound decreased in the cartilage tissue with progression of osteoarthritis. Therefore, the compound can be used to evaluate cartilage injury and repair in patients with osteoarthritis.
The present invention therefore relates to a difuro-8-pyrone of the formula I 
wherein R1 and R2 are the same or different and represent hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, thiol, amino, halogen, carboxylic acid or esters or thioesters thereof, amide, azide, imide, imine, imidazole, acetal, nitrile, nitro, sulfate, sulfonic or sulfinic acid or esters thereof, phosphate or phosphonate acids or esters thereof, silyl, sulfoxide, sulfone, oxime, phosphonate, cycloalkyl, unsaturated monocyclic hydrocarbons, aryl, and aryloxy, and salts and optically active and racemic forms of a compound of the formula I.
In an embodiment of the invention, compounds of the formula I are provided wherein R1 and R2 are the same or different and represent C1 to C6 alkyl, preferably methyl, or ethyl. Most preferably the compound of the formula I of the invention is 2,6-dimethyl-difuro-8-pyrone (also referred to herein as xe2x80x9cCartilage Marker-1xe2x80x9d or xe2x80x9cCM-1xe2x80x9d).
The invention also comprises a crystal form of a compound of the invention, a precursor of a compound of the invention, and a polymer comprising a plurality of compounds of the invention.
Further, the present invention provides a method for preparing a compound of the formula I as defined herein, and a composition, preferably a pharmaceutical composition, comprising a compound of the formula I, a crystal form or precursor thereof, or a polymer of the invention as an active agent.
Still further, the present invention provides a method of detecting a compound of the invention in a sample, comprising the step of reacting the sample with a substance that binds to, or interacts with a compound of the invention. The substance may be an antibody, which recognizes a compound of the invention.
The invention also provides a method of diagnosis of a condition involving modifications in cartilage, articular cartilage containing tissues such as intervertebral disc, trachea, and sternum, and/or cartilagenous metaplasia and neoplasia of tissues in a subject comprising the step of subjecting a biological sample from the subject to a test for detection of a compound of the invention.
The invention also provides a method of monitoring progress of a condition involving modifications in cartilage, articular cartilage containing tissues such as intervertebral disc, trachea, and sternum, and/or cartilagenous metaplasia and neoplasia of tissues in a subject, or monitoring efficacy of treatment of such a condition, comprising, periodically testing a sample from the subset for the presence of a compound of the invention.
The invention also provides a method of screening for putative therapeutic agents for the prevention and/or treatment of a condition involving modifications in cartilage, articular cartilage containing tissues such as intervertebral disc, trachea, and sternum, and/or cartilagenous metaplasia and neoplasia of tissues comprising testing the ability of the putative agents to bind to, interact with, inhibit, or stimulate a compound of the invention, or a precursor thereof. Also included are therapeutic agents identified using this method of screening.
The invention also features a method for delivering a substance to a tissue e.g. cartilage tissue, the method includes administering to a subject the substance linked to a compound of the invention. A molecular conjugate is also contemplated which includes a compound of the invention and an imaging or therapeutic agent.
The compound of the invention, and polymers thereof are planar and may be used as lubricants. For example, the compound and polymer of the invention may be used as a lubricant between cartilage.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawing. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.